PROJECT SUMMARY/ABSTRACT Polychromatic flow cytometry (FC) is one of the most powerful analytical techniques used in immunology, basic research, and clinical medicine. In basic research, FC is a primary tool for understanding disease development at the cellular and subcellular levels. In the rapidly developing field of immunotherapy, FC is an indispensable tool for monitoring the effectiveness of new therapies and identifying key cellular subpopulations using multiple biomarkers within a panel. It plays a critical role in several growing clinical applications: development of vaccines for infectious diseases, including emerging diseases such as Ebola and Zika; characterization of immunological cells and responses related to transplantation and treatment of graft-versus-host disease; and development of vaccines and immune therapies to prevent and treat HIV infections. Increasing the number of spectrally distinct fluorophores for analysis of a single sample will give researchers greater flexibility and give clinicians a higher level of accuracy in the diagnosis and management of conditions where the size of the sample use for diagnosis is limited and for specimens with low cell numbers, such as those derived from fine needle aspiration, laparoscopy, core biopsy, and cerebrospinal fluid. Due to these small sample sizes, there are often insufficient cell numbers for multiple panels. Improvements in optics and lasers have driven the recent introduction of new fluorophores for FC. However, most of these new fluorophores have broad and often overlapping emission profiles, much like traditional fluorophores. Overlapping spectra can be resolved by the combined use of bandpass filters and mathematical compensation (overlap subtraction); however such compensation increases experimental error, reduces sensitivity, and limits multiplexing. Thus, even with a prototype 50-color instrument, leading investigators have only been able to implement a 30-parameter panel ? advances in reader technology have outstripped available capabilities in fluorophores. NIRvana Sciences is currently developing two families of tetrapyrrole fluorophores licensed from NC State University. These fluorophores offer properties that make them excellent candidates for use in polychromatic FC including ultra-violet/violet excitation, long stokes shifts into the red and near infrared spectrums along with very narrow emissions. NIRvana Sciences and the Lindsey lab are developing a polymer platform that retains the intrinsic brightness of its dyes and has strong utility to the flow cytometry reagent market.